Elsevier

Experimental Eye Research

Volume 83, Issue 3, September 2006, Pages 554-559
Experimental Eye Research

Multiplex fluorescence in situ hybridization identifies novel rearrangements of chromosomes 6, 15, and 18 in primary uveal melanoma

https://doi.org/10.1016/j.exer.2006.02.007Get rights and content

Abstract

Uveal melanomas are the commonest ocular tumour of adults and are characterized by reproducible alterations of chromosomes 1, 3, 6 and 8. These alterations are of prognostic relevance and have also be shown to correlate to high risk and low risk metastatic categories of uveal melanoma as defined by micro-array analysis. It is, however, possible that a catalogue of relevant genetic alterations, involving gene rearrangement rather than amplification, have as yet eluded identification. To address this point we examined 14 primary uveal melanomas, using 24 colour multiplex fluorescence in situ hybridization (M-FISH). All tumours were karyotyped following G-Banding, and M-FISH was performed to confirm and clarify the identity of abnormal chromosomes. M-FISH data were obtained from all tumours and was able to establish the nature of most abnormalities not fully characterized by cytogenetics. Abnormalities of chromosome 6 were far more frequent than previously indicated, in approximately 70% of cases, indicating they have been substantially underrepresented in past studies of uveal melanoma. Spindle melanomas were found to have novel rearrangements affecting in particular chromosomes 6, 15 and 18, suggesting that juxtaposition of genes through translocational events may play a role in the development of some uveal melanomas. In conclusion, this study is the largest of primary uveal melanoma analysed by M-FISH and indicates that alterations of chromosome 6 have previously been underestimated. Furthermore spindle melanomas are prone to rearrangements affecting chromosomes 6, 15 and 18, which may relate to early changes in uveal melanoma development or associate with those melanomas of a more differentiated status.

Introduction

The identification of non-random chromosome changes, and a greater understanding of the complexity of chromosomal rearrangements in cancer, has been facilitated by the introduction of fluorescence in situ hybridization (FISH) and its associated methodologies. One approach is 24 colour multiplex FISH or M-FISH, which clarifies uncharacterised changes and detects alterations not found by cytogenetics (Roberts et al., 1999). Despite its value, there are relatively few primary solid tumours analysed by M-FISH, with most work performed on cell lines (Ferti et al., 2004, Naus et al., 2001, Schulten et al., 2002, Stamouli et al., 2004); a fact that reflects the technical difficulties associated with obtaining good quality chromosome preparations.

Uveal melanomas are the commonest primary ocular tumours of adults, with a highly aggressive nature; responsible for the death of approximately 50% of the patients they affect (Isager et al., 2004, Singh and Topham, 2003). Primary uveal melanomas have associated non-random chromosomal alterations (Aalto et al., 2001, Hoglund et al., 2004, Prescher et al., 1995, Sisley et al., 1990) and although there are some similarities between their genetic alterations and those of the cutaneous form, there are nevertheless striking differences (Aalto et al., 2001, Hoglund et al., 2004, Prescher et al., 1995, Sisley et al., 1990). Monosomy of chromosome 3 is seen in approximately 50% of uveal melanomas, but is a relatively infrequent event amongst cutaneous melanomas (Hoglund et al., 2004). Likewise alterations of chromosome 8 are more readily associated with uveal melanomas, but the other non-random rearrangements of chromosomes 1 and 6 found in uveal melanoma show parity with the cutaneous form (Hoglund et al., 2004). A clear relationship between abnormalities of chromosomes 3 and 8 and a poor prognosis has been demonstrated for uveal melanoma (Prescher et al., 1996, Sisley et al., 1997, White et al., 1998) and changes of chromosomes 1 and 6 may also be of value in establishing patient outcome (Kilic et al., 2005, Sisley et al., 2000, White et al., 1998). Abnormalities of chromosomes 3 and 8 are more common amongst melanomas with a ciliary body component, whilst alterations of the long arm of chromosome tend to relate to tumours derived from the choroid (Sisley et al., 2000). The existence of two independent tumourigenic pathways has been suggested (Hoglund et al., 2004, Parrella et al., 1999), and the association of chromosome changes with certain subsets of uveal melanomas, in combination with the evidence of divergence from recent micro-array data appear to confirm this premise (Onken et al., 2004, Tschentscher et al., 2003). Nevertheless until the relevant genes have been identified it remains unclear if this translates to two distinct pathways of tumourigenesis, involving entirely different sets of genes, or whether at some point there is convergence or a mixing of the pathways. It is therefore possible that as yet undisclosed genetic changes exist which could affect how uveal melanomas initiate and progress. To explore this possibility, we have used M-FISH on a series of 14 primary posterior uveal melanomas.

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Material and methods

Fourteen patients with primary posterior uveal melanoma were referred for treatment at the Academic Unit of Ophthalmology and Orthoptics during the period 2002–2003. Informed consent was obtained to use the tissues prior to removal of the tumour and protocols adhered to the principles of the Declaration of Helsinki. The clinico-pathological details of the melanomas are detailed in Table 1. Uveal melanomas were processed and established as short term cultures, and chromosome preparations were

Results

Partial karyotypes and the results of M-FISH are presented in Table 2. A representative karyotype and M-FISH from SOM 342 is presented in Fig. 1 and M-FISH of SOM 328 with a derivative 18 is shown in Fig. 2. The incidence for rearrangements of each chromosome is presented in Fig. 3.

Only one case (SOM 218) had no chromosome abnormalities detected by cytogenetic analysis, and M-FISH confirmed that discreet rearrangements were absent. In all other cases chromosome abnormalities were identified by

Discussion

To our knowledge this study represents the largest series of melanomas analysed by M-FISH, with most data being previously obtained from the analysis of cell lines (Naus et al., 2001, Schulten et al., 2002). For uveal melanoma, only a couple of primary tumours have been analysed by 24 colour, with work also on cell lines (Naus et al., 2001).

Rearrangements of chromosome 6 were by far the most frequent abnormality seen in this study, in approximately 70% of cases, making rearrangements of

Acknowledgements

This work has been supported by grants from the National Eye Research Centre (Yorkshire) (Grant No 003), and Trent Regional Health RBF00XX5.

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